Biochemical and biological assays are a primary tool utilized in many aspects of drug discovery, including fundamental research in biochemistry and biology to describe novel phenomena, analysis of large numbers of compounds, screening of compounds, clinical tests applied during clinical trials, and ultimately diagnostic tests during administration of drugs. Many biological and biochemical assays require measurement of the response of a biological or biochemical system to different concentrations of one reagent, such as an inhibitor, a substrate, or an enzyme. Typically, discrete steps of biochemical concentration are mixed within a proscribed range. The number of concentrations measured is limited by the number of dilution steps, which are limited in practice by the time and effort required to make the discrete dilutions, by the time and effort to process the resulting individual reactions, by reagent consumption as the number of reactions increases, and more strictly by pipetting errors that limit the resolution of discrete steps.
As technology advances in drug development, miniaturization and automation are active areas of innovation, with primary drivers being decreased cost (through decreased reagent use and decreased manpower) and improved data quality (through finer process control and increased process reliability). Improvements in data quality and automation frequently convey additional advantages that permit new scientific approaches to questions. Automation, if sufficiently extensive, can include software that permits automatic work scheduling to improve efficiency or statistical process control for process improvement. Again, these improvements achieve greater reliability, use less manpower, and improve throughput.
Microfluidic systems, including labs-on-a-chip (LoCs) and micro-total analysis systems (μ-TAS), are currently being explored as an alternative to conventional approaches that use microtiter plates. The miniaturization afforded by microfluidic systems has the potential to greatly reduce the amount of reagent needed to conduct high-throughput screening. Thus far, commercial microfluidic systems have shown some promise in performing point measurements, but have not been employed to mix concentration gradients and particularly continuous gradients due to technologic limitations. In particular, several challenges remain in the design of industry-acceptable microfluidic systems. Apart from cost and manufacture related issues, many sources of such challenges relate to the fact that, in a micro-scale or sub-micro-scale environment, certain fluid characteristics such as viscosity, surface tension, shear resistance, thermal conductivity, electrical conductivity, molecular diffusivity, and the like, take on a much more dominant role than other, more easily manageable factors such as weight and gravity. In addition, controlling the signal-to-noise ratio becomes much more challenging when working with nano-scale volumes and flow rates, as certain sources of noise that typically are inconsequential in macroscopic applications now become more noticeable and thus deleterious to the accuracy of data acquisition instruments.
One important consideration in the design of a microfluidic system is the means utilized for driving liquid flows. Pressure-based, electrokinetics-based, and displacement-based pumping techniques have been explored. As a general matter, pressure pumping generates a proscribed pressure difference at the two ends of a pipe. Examples of the use of pressure-driven flow in a microfluidic format, in which step-wise concentration gradients were generated in the course of enzymology-related experiments, are disclosed in Chien et al., “Multiport flow-control system for lab-on-a-chip microfluidic devices”, Fresenius J Anal Chem 371, 106-11 (2001) and Kerby et al., “A fluorogenic assay using pressure-driven flow on a microchip”, Electrophoresis 22, 3916-23 (2001).
Electrokinetic pumping techniques generally include electro-osmotic, electrophoretic, electro-wetting, and electrohydrodynamic (EHD) pumping, each of which operates on different principles than pressure and displacement pumping. For a general treatment of some types of electrokinetic pumping, see Bousse, et al., “Electrokinetically controlled microfluidic analysis systems”, Annu Rev Biophys Biomol Struct 29, 155-81 (2000).
Displacement pumping generates a proscribed flow rate directly, typically by pushing a piston or other boundary against a volume of liquid. The change in volume generated by motion of the solid boundary, therefore, is the flow rate generated by the pump. A typical example of a displacement pump is a syringe pump.
The term “displacement micropumps” has been used to describe two categories of pumps. The first category includes pumps that are themselves microscopic, and are basically miniaturized versions of macroscopic centrifugal pumps, gear pumps, peristaltic pumps, rotary pumps, and the like. Some of these pumps can be fabricated on-chip using MEMS or other microfabrication techniques, and are capable of low flow rates. However, such pumps suffer from a number of limitations: they generate pulsatile flows, and the flow rates from these pumps depend in a non-linear way upon a number of factors, including the age of the pumps, the frequency with which the pumps are “pulsed”, and their precise location on a chip. These factors make it difficult to use such pumps to achieve reliable and reproducible flow rates of the sort necessary to achieve controlled gradients. Additionally, these pumps are fabricated with semiconductor and MEMS manufacturing techniques. This fabrication can be extremely costly and time-consuming, and results in a specific pump-architecture that is not flexible or reconfigurable and, frequently, is not manufacturable according to industry-acceptable considerations.
The second category of displacement micropumps includes macroscopic pumps that are capable of delivering microscopic flow rates. Again, there are a wide variety of such pumps available. Some micropumps have minimum flow rates of tens of microliters per minute. Unfortunately, a μl/min-scale flow rate is three orders of magnitude larger than the nl/min-scale flow rates often desired by researchers interested in microfluidics-based assays and experiments, and nl/min flow rates have heretofore been unattainable with these pumps. The pumps that are of primary interest in this category are so-called syringe pumps. A syringe pump typically consists of a motor connected to, for example, a worm gear that pushes the plunger of a syringe, causing liquid to flow out of the syringe tip. The syringe is often coupled to whatever device or instrument requires the flow. Syringe pumps designed for low flow rates are commercially available. Some of these pumps are capable of delivering μl/min-scale flow rates. Most of these pumps, however, use stepper motors, which become unacceptably pulsatile as the step rate is decreased to drive very slow flows. While some syringe pumps use servomotors, they are not capable of practicing stable, precise, controllable flow rates below the μl/min scale. For many applications, such as dispensing predefined aliquots of liquid, pulsatile flows are acceptable. However, when a linear, or smoothly varying, continuous gradient is desired, the quality of flow from pumps utilizing stepper motors decreases as the flow rate drops, adding noise to the gradient at the extremes of the gradient. In contrast, a servomotor is capable of moving at any speed (in non-discrete steps), because the rotation rate is directly controlled (not the frequency of steps).
Another factor in the design of microfluidic systems is the microfluidic interconnect, which generally provides a fluidic interface between a microfluidic component and either another microfluidic component or a macrofluidic component. As with all fluidic connections, a microfluidic interconnect should create a mechanically stable, fluid-tight connection between the components that can contain the pressures of the fluids. Additionally, a microfluidic interconnect should have a small dead volume so as not to approach or exceed the volume of the microfluidic device associated therewith. Moreover, dead volumes should be kept small for the sake of efficiency because, by nature, a sample is neither prepared nor analyzed in a dead volume. In addition, a microfluidic interconnect should not have outpockets, create rapid expansions of channel volumes, or introduce sharp turns, so that the interconnect does not generate excessive dispersion of chemical concentration gradients. The interconnect should not trap bubbles because this affects the accuracy of displacement flow rates, and, as a result, affects time of flight and concentration. Finally, the interconnect should be manufacturable in a precise, reliable, and repeatable manner.
One consideration when employing a microfluidic system to acquire data is thermal noise. For example, room temperature fluctuations can influence flow rates and measurements of the flows and of chemical reactions. There are several reasons that temperature fluctuations cause noise. Among other things, the fluorescent dyes often utilized to monitor reaction rates are pH dependent, and many pH buffers are temperature dependent. The rates of reaction of enzymes are strongly temperature dependent. Also, physical changes to components in the system due to thermal expansion can affect flows and measurements. Thermal changes in the fluid paths can change flow characteristics, flow rates and fluid velocities. For example, a change of only 0.01% volume over 1 minute for a volume of 10 microliters equals a volume change of 1 nl, which is problematic if flows of 1 nl/min are being studied. When trying to control flow rates of nl/min, very small changes in volume can produce significant changes in the observed flows. Thermal changes in the alignment of components, similarly, can have undesired effects owing to the small sizes of microfluidic components. For example, consider a photodetector that has been positioned to perform optical measurements in the center of a microfluidic channel that is 10 μm wide. Thermal expansion of only a few micrometers can move the photodetector off-center or even entirely away from the channel. Similarly, many microfluidic chips are made of bonded or laminated materials. These laminated structures are highly prone to flexing due to thermal expansion of the laminates, especially if one laminate expands more than another. This flexing of the chip can change the position of a microchannel that has been, for example, positioned into the beam of a laser for photo-measurement of a chemical reaction in the channel.
The embodiments described herein are provided to address these and other problems attending current microfluidic systems.